Vitality and metabolic properties of binucleate and trinucleate pollen species upon dehiscence

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Chapter 1.

Effects of various components upon germination in vitro were studied in order to develop an optimal germination medium for Compositae pollen. Equilibration of pollen in humid air, preceding germination, improved the reliability of results considerably.

Irregular germination ability of pollen samples, originating from different collections, was studied by exposing flowering plants to different climatic conditions. High relative humidity and temperature at dehiscence cause a rapid decrease in pollen vitality. Data for an optimal germination medium and for acquisition of good pollen quality are presented.

Chapter 2.

The respiration and vitality of ungerminated bi- and trinucleate pollen were studied in order to determine the influence of relative humidity and temperature on metabolic activity. The gas exchange, gerniination capacity and staining with tetrazolium bromide were followed under standardized conditions.

A constant respiration rate occurred under conditions of high relative humidity (97 %). Per mg pollen, the trinucleate grains of Compositae and Gramineae respired 2 to 3 times as intense as 6 species of binucleate grains. Per unit of pollen protein the differences were even larger. In contrast to binucleate pollen, the longevity of trinucleate pollen was very short and the ability to germinate was lost twice as fast as the respiration capacity. This limits the use of tetrazolium bromide as an indicator of viability.

At reduced relative humidities respiration was strongly restricted, but the longevity of bi- and trinucleate pollen considerably increased.

Pollen of Gramineae, however, was very sensitive to changes in relative humidity; short exposure to low relative humidity decreased both the vitality and the capacity to respire.

Chapter 3.

Bi- and trinucleate pollen generally differ in the extent of their mitochondrial development at anther dehiscence and in the rate of their attainment of maximum-phosphorylative capacity during germination in vitro, as judged from experiments with representatives of both groups.

The typically trinucleate pollen of Aster tripolium L. immediately respired at a high rate, maintaining a high energy charge. Mitochondria attained maximum electron-transducing capacity within 2 min of incubation, while tube growth started within 3 min. In contrast, the binucleate pollen of Typha latifolia L. only gradually reached a relatively low rate of respiration, concomitant with a temporary decrease in energy charge, upon immersion in the germination medium. Development of the mitochondrial, electron-transducing system occurred in about 75 min, after which the first pollen tubes emerged. Starting from a poor differentiation, mitochondria became increasingly normal in appearance as germination proceeded.

The binucleate pollen of Nicotiana alata Link et Otto and Tradescantia paludosa Anders. et Woods. showed intermediate characteristics: Nicotiana resembled Typha but mitochondria developed at a higher rate; Tradescantia germinated more rapidly and resembled the trinucleate pollen of Aster..

Inhibitors of mitochondrial or cytoplasmic protein synthesis failed to affect the development of the mitochondrial, respiratory capacities during pollen germination. It is concluded that the duration of the lag period is determined by the level and rate of mitochondrial development and not by the division of the generative cell.

Chapter 4.

The equal rates of water vapour absorption by both bi- and trinucleate pollen indicate that their widely-differing rates of respiration have an intrinsic, biochemical basis. This was investigated with various metabolic inhibitors that were previously introduced into dry pollen via anhydrous acetone.

The uncoupler, CCCP, inhibited the O 2 uptake of rapidly respiring pollen and stimulated that of slowly respiring types to similar absolute values, that probably reflect the rates of substrate transport across the mitochondrial membranes.

The extent of inhibition of the O 2 uptake by oligomycin, DCCD, antimycin A, and SHAM, alone and in combinations, indicates that hardly any oxidative phosphorylation and anabolic activities occur in slowly respiring, binucleate pollen species, having low-developed mitochondria and high EC values. The presence of the alternative pathway was insignificant.

In other binucleate pollen species, characterized by recognizable mitochondria and low EC values, a limited ATP synthesis was established. The low EC values point to imbalance between phosphorylative and anabolic activities.

In rapidly respiring, trinucleate pollen, containing well- developed mitochondria, a significant activity of the alternative oxidase was found. The EC values were high notwithstanding the large demand for ATP, mounting to 1.7 micro-mol h -1mg pollen -1.

In some pollen species, oligomycin highly stimulated the flow of electrons through the cytochrome pathway, which made an estimation of the ATP synthesis impossible.

Chapter 5.

Under humid conditions both bi- and trinucleate pollen species incorporate very low amounts of leucine, 0.4 pmol min -1mg pollen -1on an average. During germination in vitro , however, the two types of pollen greatly differ in their capacity for protein synthesis.

Binucleate pollen species such as Typha , which are characterized by slow respiration in humid air and prolonged lag periods during germination in vitro , contain large amounts of monoribosomes at dehiscence. Polyribosomes are formed soon after the pollen is wetted in the germination medium and a considerable incorporation of leucine is initiated after 10-15 min.

More rapidly respiring, binucleate pollen, such as Tradescantia , showing a short lag period, may contain many polysomes at dehiscence already and incorporates leucine within 2 min of incubation.

On the contrary, rapidly respiring, trinucleate Compositae pollen contains very limited amounts of ribosomal material and never attains any substantial level of incorporation.

Cycloheximide completely inhibited both protein synthesis and tube emergence and growth in the slowly respiring binucleate pollen species. The more rapidly respiring types are less dependent on protein synthesis, while germination of the phylogenetically advanced, trinucleate Compositae pollen proceeds completely independently.

It is concluded that the level of phylogenetic advancement of the male gametophyte is characterized by its overall state of metabolic development at dehiscence rather than by the number of its generative cells.

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